A healthy retina is necessary for good vision. Retinal disorders can cause partial or total loss of vision. Many retinal diseases share common symptoms and treatments, but each has unique characteristics. The goal of retinal disease treatments is to stop or slow disease progression and preserve or restore loss vision.
The neuroretina is a complex neurological tissue composed of a network of eight interconnected cell layers responsible for transforming visual light into electromechanical information that is sent to and interpreted by the brain through the optic nerve. The arrangement of the neural cells within the retina requires light to travel through most cell layers to reach the photoreceptors located in the posterior part of the retina; the photoreceptors thereafter transmit information to retinal neurons for local processing of visual information and transmission to the visual cortex. There are two types of photoreceptors, rods and cones. Both types of photoreceptors are present throughout the retina but rods dominate the periphery whereas cones are most dense in the center of the retina. The center of the retina, also known as macula, is a specialized region of the retina with densely packed cones and high concentration of pigments where vision is most acute. One of the main characteristics of the retina is its transparency. The transparency allows light to reach the outermost layer of the retina where the photoreceptors are located. This transparency requirement implies that the vasculature needed to nourish and support the retina is extremely specialized. Blood supply to the retina is provided by to main sources: the retinal vasculature and the choroid. The choroid is a highly vascularized, pigmented tissue lying between the retina and the sclera. The choroid provides nutrients, metabolites and gaseous exchange to the retina by diffusion through chorio-capillaries. The retinal pigment epithelium (RPE) is a monolayer of pigmented cells situated between the neuroretina and the choroid. RPE cells protect, support, and feed the light sensitive retina. The particular environment of the neuroretina is maintained by the blood-retinal barrier (BRB), also called hemato-retinal barrier. The BRB is constituted by the inner blood-retinal barrier and the external blood-retinal barrier. The inner blood-retinal barrier is formed by the tight junctions between capillary endothelial cells of the retinal vasculature. The external blood-retinal barrier is constituted by the tight junctions of RPE cells. Tight junctions between RPE cells are essential to control the transport of liquid and soluble compounds through the BRB, as well as to avoid entrance of toxic substances into the retina.
Blood vessels are formed in the retina by two major processes: vascularization or angiogenesis. Vascularization occurs as a result of differentiation of precursor cells, which are already present in the tissue, into the endothelial cells that contribute to the formation of blood vessels. Angiogenesis differs in that the new blood vessels are generated by sprouting from the preexisting vasculature. Angiogenesis requires proliferation, migration and differentiation of endothelial cells; as well as maturation of the newly formed vessels. The number of endothelial cells is normally stable in an adult organism; the stability in endothelia is controlled by a balance in the concentration of angiogenic and anti-angiogenic factors.
Alterations in the balance of factors lead to induction or suppression of angiogenesis. Vascularization and angiogenesis are natural processes that take place during development and other events such as healing; but these processes also have a role in the pathogenesis of certain diseases. Pathological neovascularization usually implies a combination of both vascularization and angiogenesis. There are two types of neovascularization that occur in the retina and both can cause vision loss: retinal neovascularization (RNV) in which new vessels sprout from the retinal capillaries and invade the vitreous and neural retinal layers, and choroidal neovascularization (CNV) in which new vessels sprout from the choroidal vasculature and invade the subretinal space. Although RNV and CNV originate from different vascular networks and invade different layers of the retina, shared molecular mechanisms promote the progression of both. RNV and CNV are the most common causes of severe visual loss in developed countries and new treatments are needed.
Vascular endothelial growth factor (VEGF), one of the most important mediators of angiogenesis, is upregulated during RNV and CNV. Over the last decade, scientists have developed several new “anti-VEGF” drugs. They help block abnormal blood vessels, slow their leakage, and help reduce vision loss. Treatment with anti-VEGF drugs is performed by intravitreal injections.
Intravitreal (IVT) injection is the most common method for delivering drugs to the back of the eye, which is used by all the currently approved drugs for the treatment of retinal disease with exception of verteporfin. Verteporfin is administered by intravenous injection followed by laser treatment, but its use has significantly decreased due to the marketing of the modern anti-VEGF treatments. The reasons behind the extended use of IVT injection are efficiency delivering drugs, level of familiarity to retinal physicians and ability of the physician to control treatment compliance (Rowe-Rendleman et al 2014). This method comes however with its own set of very specific disadvantages that include patient discomfort, risk of endophthalmitis, cataract formation and retinal detachment as well as high associated cost due to the office-based administration. Other methods of administration include periocular injection, suprachoroidal injection, sub-tenon injection and also eye drops. However, there is a certain scepticism about whether sufficient efficacy can be achieved to treat retinal conditions with eye drops, since the active ingredient has to be delivered from the cornea to its site of action in the retina. There are significant barriers and eliminations pathways that hinder the delivery of drugs to the back of the eye. Firstly only 1-7% of the administered drug is absorbed by the eye; most of the drug administered as eye drops is drained out of the eye or absorbed via the nasolacrimal duct to systemic circulation. In addition, drugs are rapidly cleared from the vitreous humor. There are two routes of clearance from the posterior cavity, the anterior and the posterior. The former entails clearance to the anterior chamber by the aqueous humor (AH) flow and thereafter by the AH outflow through the anterior chamber angle. The latter implies elimination through the blood-retinal barrier. Thus, drugs that can easily permeate through the blood-retinal barrier would have a very short half-life in the vitreous humor.
An alternative to anti-VEGF drugs for the treatment of retinal diseases related to neovascularization are RNA interference (RNAi) based drugs.
RNAi is a naturally occurring post-transcriptional regulatory mechanism present in most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans {Fire et al 1998} was awarded the Nobel Prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells by means of double-stranded small interfering RNAs (siRNAs) 21 nucleotides long {Elbashir et al 2001}.
The process of RNA interference is thought to be an evolutionarily-conserved cellular defence mechanism used to prevent the expression of foreign genes and is commonly shared by diverse phyla and flora, where it is called post-transcriptional gene silencing. Since the discovery of the RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise “undruggable” with traditional pharmaceutical approaches involving small molecules or proteins.
According to current knowledge, the mechanism of RNAi is initiated when long double stranded RNAs are processed by an RNase III-like protein known as Dicer. The protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) {Collins et al 2005} and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3′ overhangs and a 5′ phosphate and 3′ hydroxyl group. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences {Elbashir et al 2001} upon adenosine-triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity {Nykanen et al 2001}. The catalytic activity of RISC, which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AGO2) {Liu et al 2004; Song et al 2004}. AGO2 belongs to the highly conserved Argonaute family of proteins. Argonaute proteins are ˜100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains {Cerutti et al 2000}. The PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs. AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5′ end {Elbashir et al 2001}. An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex {Rand et al 2005}. Crystallography studies analyzing the interaction between the siRNA guide strand and the PIWI domain reveal that it is only nucleotides 2 to 8 that constitute a “seed sequence” that directs target mRNA recognition by RISC, and that a mismatch of a single nucleotide in this sequence may drastically affect silencing capability of the molecule {Ma et al 2005; Doench et al 2004; Lewis et al 2003}. Once the mRNA has been cleaved, due to the presence of unprotected RNA ends in the fragments the mRNA is further cleaved and degraded by intracellular nucleases and will no longer be translated into proteins {Orban et al 2005} while RISC will be recycled for subsequent rounds {Hutvagner et al 2002}. This constitutes a catalytic process leading to the selective reduction of specific mRNA molecules and the corresponding proteins. It is possible to exploit this native mechanism for gene silencing with the purpose of regulating any gene(s) of choice by directly delivering siRNA effectors into the cells or tissues, where they will activate RISC and produce a potent and specific silencing of the targeted mRNA. RNAi has been applied in biomedical research such as treatment for HIV, viral hepatitis, cardiovascular and cerebrovascular diseases, metabolic disease, neurodegenerative disorders and cancer {Angaji S A et al 2010}.
Many studies have been published describing the ideal features a siRNA should have to achieve maximum effectiveness, regarding length, structure, chemical composition, and sequence. Initial parameters for siRNA design were set out by Tuschl and co-workers in WO02/44321, although many subsequent studies, algorithms and/or improvements have been published since then. siRNA selection approaches have become more sophisticated as mechanistic details have emerged, in addition further analysis of existing and new data can provide additional insights into further refinement of these approaches {Walton S P et al 2010}. Alternatively, several recent studies reported the design and analysis of novel RNAi-triggering structures distinct from the classical 19+2 siRNA structure and which do not conform to the key features of classical siRNA in terms of overhang, length, or symmetry, discussing the flexibility of the RNAi machinery in mammalian cells {Chang C I et al 2011}.
Also, a lot of effort has been put into enhancing siRNA stability as this is perceived as one of the main obstacles for therapy based on siRNA, given the ubiquitous nature of RNAses in biological fluids. Another inherent problem of siRNA molecules is their immunogenicity, whereby siRNAs have been found to induce unspecific activation of the innate immune system. The knockdown of unintended genes (mRNAs) is a well-known side effect of siRNA-mediated gene silencing. It is caused as a result of partial complementarity between the siRNA and mRNAs other than the intended target and causes off-target effects (OTEs) from genes having sequence complementarity to either siRNA strand. One of the main strategies followed for stability enhancement and OTE reduction has been the use of modified nucleotides such as 2′-O-methyl nucleotides, 2′-amino nucleotides, or nucleotides containing 2′-O or 4′-C methylene bridges. Also, the modification of the ribonucleotide backbone connecting adjacent nucleotides has been described, mainly by the introduction of phosphorothioate modified nucleotides. It seems that enhanced stability and/or reduction of immunogenicity are often inversely proportional to efficacy {Parrish, 2000}, and only a certain number, positions and/or combinations of modified nucleotides may result in a stable and non-immunogenic silencing compound. As this is an important hurdle for siRNA-based treatments, different studies have been published which describe certain modification patterns showing good results, examples of such include EP1527176, WO2008/050329, WO2008/104978 or WO2009/044392, although many more may be found in the literature {Sanghvi Y S. 2011; Deleavey et al 2012}.
The eye is a relatively isolated tissue compartment, which provides advantages for utilization of siRNA-based drugs for treating retinal diseases related to neovascularization. Feasibility of using siRNA for treatment of CNV has been demonstrated using siRNAs administered by intravitreal injection directed against VEGF or VEGF receptor 1 (VEGFR1) {Campochiaro P A. 2006}. Delivery of siRNAs by topical instillation to the posterior segment is truly challenging, because of the relatively large distance that the siRNAs have to go through the vitreous body before they reach the retina {Guzman-Aranguez A. et al 2013}. In addition, pharmaceutical treatment of retinal diseases affecting the posterior segment of the eye is also made challenging by restrictive blood ocular barriers such as the blood aqueous barrier (BAB) and the BRB, which separate the eye from systemic circulation. Furthermore, the compartmentalized structure of the eye limits the passage of siRNAs from the anterior chamber to the posterior segment of the eye {Duvvuri S et al 2003}. Finally, once siRNAs successfully enter the back of the eye, effective clearance mechanisms act to rapidly clear the delivered molecules {Del Amo E M et al 2008}. Thus, direct injection into the vitreous cavity has become the most efficient means to deliver siRNA-based therapeutics into the posterior segment of the eye {Edelhauser H F et al 2010}. Intravitreous injection of siRNAs achieves high concentrations of siRNAs that are locally available to the retinal tissues while limiting systemic exposure. However, the concentration of siRNAs is rapidly depleted from the posterior segment due to degradation by vitreous endonucleases and/or via permeation across the BRB and by diffusion across the vitreous to the anterior chamber. Thus, multiple intravitreal injections are required to maintain optimal siRNA concentrations within the posterior segment of the eye. The main disadvantage of this administration mode is that multiple intravitreal injections are associated with raised intraocular pressure, vitreous or retinal hemorrhage, retinal detachment, retinal tears, endophthalmitis, cataracts, floaters and transient blurry vision {Edelhauser H F et al 2010}. Therefore, while intravitreal injections ensure delivering a high concentration of siRNA to the retina, this method of administration also comes with its own set of particular risks. Consequently, topical administration of siRNAs could reduce risks and entail a more patient-friendly method of administration.
Naked siRNAs have shown to reach certain regions following topical applications, but access to deeper regions such as the innermost layer of the retina and effective cellular uptake require the development of strategies that ensure sufficient concentration of the compound reaching the cytoplasm of cells located in the target area and provoke a desired physiologic or therapeutic response. Physical approaches to deliver siRNAs across the stratum corneum barrier include microneedles (Chong, Gonzalez-Gonzalez et al., 2013), intradermal injection (Leachman, Hickerson et al., 2010), electroporation (Nakai, Kishida et al., 2007), iontophoresis (Kigasawa, Kajimoto et al., 2010) among others. Modifications of the molecule and/or formulation can also enable the molecule to penetrate into the required region and improve cellular uptake.
Topical administration of siRNA-based therapeutics for the treatment of retinal diseases has been described; for instance, US20130123330 discloses the treatment of diabetic retinopathy and other ocular neovascularization diseases by administering at least a siRNA duplex binding to mRNA molecules encoding VEGF or VEGFR2, or a cocktail combining siRNA duplexes targeting both genes VEGF and VEGFR2. This patent application described that the siRNA duplexes may be administered to the eye topically, subconjunctivally, or intravitreally. However, the specification only includes examples of compounds administered intravitreally or subconjunctivally. WO2010048352 (Quark Pharmaceuticals) discloses the use of chemically modified siRNA compounds for the treatment of ocular diseases, disorders and injuries associated with degeneration or death of retinal ganglion cells, including retinitis pigmentosa (RP), diabetic retinopathy (DR), diabetic macular edema (DME) and age related macular degeneration (AMD). Although the topical delivery to retinal tissue has been demonstrated for siRNA compounds which down-regulate the expression of target genes associated with loss of these cells, such as CASP2, RTP801, TIGASEII and p53 genes, only siRNA compounds targeting Caspase 2 have been proven to provide an ocular neuroprotective effect by increasing the survival of the retinal ganglion cells.
Target gene selection plays a key role when treating and/or preventing retinal diseases related to neovascularization with siRNA-based therapeutics. Notch-regulated ankyrin repeat protein (NRARP), is induced by Notch at newly formed branch points, where it differently modulates Notch and Wnt signaling activity to balance stalk proliferation and vessel stability. siRNA mediated downregulation of NRARP in HUVECs correlates with an increase in Notch, which in stalk cells is translated to vessel regression whereas increased Notch leads to formation of new tip-cells {Phng L K, Potente M, et al. 2009}. Therefore, it is likely that NRARP plays an important role in the regulation of the angiogenesis and/or neovascularization processes in retinal tissues.
siRNA-based therapeutics can slow down and prevent the progression of RNV and CNV in retinal diseases, but the therapeutic benefits can be diminished by inefficient siRNA delivery and the limited duration of siRNA bioavailability, which requires prolonged treatment regimens of repeated intravitreal injections. Thus, improved and non-invasive siRNA-based therapeutics targeting new and inventive target genes must be designed for the treatment and/or prevention of retinal diseases related to neovascularization.